Molecular Cloning And Transformation Of Sedoheptulose-1, 7-Bisphosphatase In Lycopersicon Esculentum

Tomato (Lycopersicon esculentum) is an important cold-sensitive vegetable cultivated in solar-greenhouse. Sedoheptulose-1, 7-bisphosphatase is a key enzyme in the photosynthetic carbon reduction cycle, known as Calvin cycle. SBPase plays a crucial role in the regeneration of ribulose-1, 5-bisphosphate (RuBP) and the control of carbon flux because of its location at the branch point of RuBP regeneration and starch biosynthesis. Thus, it is promising to improve the photosynthesis by increasing the level and activity of SBPase through genetic engineering.In the present study, we cloned the SBPase gene from tomato plant and analyzed the SBPase expression in different tissues as well as diurnal expression patterns. Sense and anti-sense constructs were made and used for agrobacterium- mediated transformation of tomato plants. The transgenic plants were utilized to analyze the effects of SBPase changes on the growth and photosynthesis in tomato plants. The results of the present research were as follows:1. The full-length cDNA encoding SBPase (designated as LeSBP, GenBank accession No. FJ959073) was isolated from tomato leaves by RT-PCR and RACE. The cDNA contained 1,564 nucleotides with a complete open reading frame (ORF) of 1,185 nucleotides, which encoded a peptide of 394 amino acids, with an estimated molecular mass of 42 kDa. SBPase amino acid sequence had high homology to those from other plant species.2. Sequence alignment revealed that LeSBP has 83.80 %, 80.71 %, 80.05 %, 78.45 % and 76.65 % similarities to SBPase amino acid sequences of Cucumis sativus, Spinacia oleracea, Arabidopsis thaliana, Oryza sativa and Triticum aestivum, respectively. Sequence comparison with SBPase from other plant species showed six conservative Cys (cysteine), and Cys 3 and Cys 4 form a CGGT(A/Q) C motif, which is believed to participate in light-regulated reaction.3. A recombinant of prokaryotic expression vector pET-LeSBP was constructed and transformed to E. coli. BL21, and expression was induced with IPTG. The strong induced fusion protein was used to immunize BALB/c male mice to obtain primary antibody. Blood was collected and the sera were tested with an enzyme-linked immunosorbent assay. The results showed that the antibody obtained was qualified for tomato SBPase protein analysis.4. The expression of SBPase in tomato plants investigated by real-time RT-PCR and western blot analyses showed that SBPase was detected in leaves, stems and fruits, but was not observed in roots. SBPase was more abundant in leaves than in other organs. The results also revealed that SBPase expression was higher in mid-position leaves than in base position leaves and upper position leaves. Measurements of Pn showed that the Pn of 8th apical leaves was higher than that of 4th, 12th, 1st and 16th leaves and the similar trend was observed in the changes of SBPase activity. The diurnal variations of gene expression at the mRNA level and SBPase activity in optimal functional leaf (8th leaf) was was similar to that of Pn, indicating the close relationship between SBPase and photosynthesis in tomato plants.5. Sense and anti-sense constructs were made by subcloning the full-length SBPase cDNA into the expression vector pBI121. The resulting plasmid was used for agrobacterium-mediated transformation of tomato plants. The successful transformants were screened and confirmed by real-time PCR and western blot. The sense transgenic plants showed the enhanced level of SBPase, while the antisense plants had decreased SBPase.6. Overexpression of SBPase led to increased SBPase mRNA abundance and activity. Compared with wild-type tomato plants, sense transgenic plants showed the enhanced Pn, increased accumulation of carbohydrate, widened stem, stronger root system, enlarged leaves and earlier flowering time. However, antisense suppression of SBPase in tomato plants led to the contrary changes to those by overexpression.7. SBPase expression in tomato plants was inhibited by low temperature stress, resulting in decreased mRNA level and enzyme activity, which significantly reduced the net photosynthesis and insignificantly affected the Fv/Fm andфPSII. In comparison with wild-type tomato plants, SBPase over-expressing plants had less reduction in Pn and EC under low temperature conditions, however, these plants showed no significant changes in Fv/Fm andфPSII. Anti-sense suppression of LeSBP led to significantly decreased Pn and increased EC in tomato plants. Our results showed that over-expression of SBPase increased Pn and improved the cold tolerance in tomato plants under low temperature stress.